Single molecule MATAC-seq reveals key determinants of DNA replication origin efficiency

Stochastic origin activation gives rise to significant cell-to-cell variability in the pattern of genome replication. The molecular basis for heterogeneity in efficiency and timing of individual origins is a long-standing question. Here, we developed Methylation Accessibility of TArgeted Chromatin domain Sequencing (MATAC-Seq) to determine single-molecule chromatin accessibility of specific genomic loci after targeted purification in their native chromatin context. Applying MATAC-Seq to selected early-efficient (EE) and late-inefficient (LI) budding yeast replication origins revealed large heterogeneity of chromatin states. Disruption of INO80 or ISW2 chromatin remodeling complexes leads to changes at individual nucleosomal positions that correlate with changes in their replication efficiency. We found a chromatin state with an optimal 100-115bp nucleosome-free region in combination with surrounding well-positioned nucleosomes and open +2 linker region is a strong predictor for efficient origin activation. Thus, MATAC-Seq identifies the large spectrum of alternative chromatin states that co-exist on a given locus previously masked in population-based experiments and provides a mechanistic basis for origin activation heterogeneity during DNA replication of eukaryotic cells. Consequently, our single-molecule assay for chromatin accessibility will be ideal to define single-molecule heterogeneity across many fundamental biological processes such as transcription, replication, or DNA repair in vitro and ex vivo. Overall design: Nanopore sequencing analysis of chromatin accessibility using DNA methylation footprinting of specific chromosomal domains containing selected replication origins that were purified by a site-specific recombination and affinity purification system